Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. We undertook to identify the unknown photopigment that mediates photo-entrainment, the process by which light resets the endogenous clock, in collaboration with Mark Rollag and Ignacio Provencio (USUHS), and Maribeth Eiden (NIMH). We have made retroviral vectors carrying sense and antisense versions of candidate photopigments to test the effects of over- and under-expression of these proteins on the responses of chick pineal cells to light. Melanopsin and pinopsin are each novel photopigments present in chick pineal cells and the best candidates for the photopigments mediating photoentrainment. Iodopsin, a photopigment used for color vision in the retina, but absent from the pineal, provides a control. Last year, we isolated the cDNAs for melanopsin, pinopsin, and iodopsin, inserted them into plasmids, and prepared retroviral vectors containing these genes. We also determined the endogenous levels of gene expression for each photopigment gene and its tissue distribution using quantitative PCR. This year, we demonstrated transduction of each gene by its respective retroviral vector. We showed increases in melanopsin protein by immunoblot and immunocytochemistry, but have not yet developed a suitable assay for determination of endogenous levels. Pinopsin antibodies have not been fully tested. We have also made probes and constructs for the per2, cry1, and cry2 genes in order to seek the point of entry of photoentraining signals into the clock. We also undertook to determine whether activation or inhibition of MAPK pathways are necessary or sufficient for photoentrainment. This project has been completed. We found that the effects of agents on levels of activated MAPK do not correlate with their effects on the circadian clock. We also found a remarkable new effect: Changing the medium is sufficient to induce large , rapid, and transient increases in levels of activated MAPK. Whether the medium is changed or not (at times that light and other agents induce phase shifts) made no difference to the phase of the melatonin rhythm in subsequent cycles. With appropriate controls, we also found that light pulses, which prominently induce phase shifts, had no effect on activated MAPK levels. Finally, to be sure that our measurements of activated MAPK levels have reflected events in the pinealocytes themselves, we used immunocytochemistry with antibodies against the pinealocyte specific enzyme HIOMT and against total MAPK, respectively, to determine the proportion of cells in the culture that are indeed pinealocytes. Results showed that pinealocytes comprise the major cell type in the cultures. Taken together, the evidence strongly indicates that changes in activation of MAPK are neither necessary nor sufficient for entrainment of the circadian clock in these cells.